Investigation of the role of protein kinase C (PKC) in cytostasis induced by tumour promoting phorbol esters and related compounds

Protein kinase C (PKC) is considered to be the major receptor for tumour promoting phorbol esters such as 12-0- tetradecanoylphorbol-13-acetate (TPA). These agents evoke a plethora of biological effects on cells in culture. The growth of A549 human lung carcinoma cells maintained in medium fortified with 10% foetal calf serum (FCS) is arrested for 6 days by TPA and other biologically active phorbol esters. In the work described in this thesis, the hypothesis was tested that modulation of PKC activity is closely related to events pivotal for cytostasis to occur. The effect of several phorbol esters, of newly synthesized analogues of diacylglycerols (DAG) and of bryostatins (bryos) on cell growth and ability to modulate activity of PKC has been investigated.Determination of the subcellular distribution of PKC following treatment of cells with TPA and partial enzyme purification by non-denaturing poly-acrylamide gel electrophoresis revealed translocation of enzyme activity from cytosoUc to paniculate fraction. Chronic exposure of cells to TPA resulted in a time and concentration dependent degradation of enzyme activity. Synthetic DAG and DAG analogues, unable to arrest the growth of cells at non-toxic concentrations, were neither able to affect subcellular PKC distribution nor compete effectively for phorbol ester binding sites at physiologically relevant concentrations. Bryos 1,2,4 and 5, natural products, possessing antineoplastic activity in mice, elicited transient arrest of A549 cell growth in vitro. They successfully competed for phorbol ester receptors in A549 cells with exquisite affinity and induced a shift in sub-cellular PKC distribution, though not to the same extent as PTA. Enzyme down-regulation resulted from prolonged exposure of cells to nanomolar concentrations of bryos. In vivo studies demonstrated that neither PDBu nor bryo 1 was able to inhibit A549 xenograft growth in athymic mice. The growth of A549 cell populations cultured under conditions of serum-deprivation was inhibited only transiently by biologically active phorbol esters. Fortification of serum-free medium with EGF or fetuin was able to partially restore sensitivity to maintained growth arrest by PTA. PKC translocation to the paniculate cellular fraction and subsequent enzyme down-regulation, induced by TPA, occurred in a manner similar to that observed in serum-supplemented cells. However, total PKC activity and cytosolic phorbol ester binding potential were greatly reduced in the serum-deprived cell population. Western blot analysis using monospecific monoclonal antibodies revealed the presence of PKC-a in both A549 cell populations, with significantly reduced protein levels in serum- deprived cells. PKC-/9 was not detected in either cell population.

Investigation of the antiproliferative properties of tumour promoting phorbol esters and related compounds

Tumour promoting phorbol esters such as 12-0-tetradecanoylphorbol-13-acetate (TPA) exert a multitude of biological effects on many cellular systems, many of which are believed to be mediated via the activation of the enzyme protein kinase C (PKC). TPA and other biologically active phorbol esters inhibited the proliferation of the A549 human lung carcinoma cell line. However, after 5-6 days culture in the continued presence of the phorbol ester cells began to proliferate at a rate similar to that of untreated cells. Resistance to TPA was lost following subculturing, although subculture in the presence of 10 nM TPA for more than 9 weeks resulted in a more resistant phenotype. The selection of a TPA-resistant subpopulation was not responsible for the observed resistance. The antiproliferative properties of other PKC activators were investigated. Mezerein induced the same antiproliferative effects as TPA but synthetic diacylglycerols (DAGs), the presumed physiological ligands of PKC, exerted only a non-specific cytotoxic influence on growth. Bryostatins 1 and 2 were able to induce transient growth arrest of A549 cells in a manner similar to phorbol esters at nanomolar concentrations, but at higher concentrations blocked both their own antiproliferative action and also that of phorbol esters and mezerein. Fourteen compounds synthesized to mimic features of the phorbol ester pharmacophore and/or DAGs did not mimic the antiproliferative properties of TPA in A549 cells and exerted only a DAG-like non-specific cytotoxicity at high concentrations. The subcellular distribution and activity of PKC was determined following partial purification by non-denaturing polyacrylamide gel electrophoresis. Treatment with TPA, mezerein or bryostatins resulted in a concentration-dependent shift of PKC activity from the cytosol to cellular membranes within 30 min. Significant translocation was not observed on treatment with DAGs. Chronic exposure of cells to TPA caused a time- and concentration dependent down-regulation of functional PKC activity. A complete loss of PKC activity was also observed on treatment with growth-inhibitory concentrations of bryostatins. No PKC activity was detected in cells resistant to the growth-inhibitory influence of TPA. Measurement of intracellular Ca2+ concentrations using A549 cells cultured on Cytodex 1 microcarrier beads revealed that TPA, mezerein and the bryostatins induced a similar rapid rise in intracellular Ca2+ levels.

Tear analysis and lens-tear interactions:part II. Ocular lipids-nature and fate of meibomian gland phospholipids

Purpose: Published data indicate that the polar lipid content of human meibomian gland secretions (MGS) could be anything between 0.5% and 13% of the total lipid. The tear film phospholipid composition has not been studied in great detail and it has been understood that the relative proportions of lipids in MGS would be maintained in the tear film. The purpose of this work was to determine the concentration of phospholipids in the human tear film. Methods: Liquid chromatography mass spectrometry (LCMS) and thin layer chromatography (TLC) were used to determine the concentration of phospholipid in the tear film. Additionally, an Amplex Red phosphatidylcholine-specific phospholipase C (PLC) assay kit was used for determination of the activity of PLC in the tear film. Results: Phospholipids were not detected in any of the tested human tear samples with the low limit of detection being 1.3 µg/mL for TLC and 4 µg/mL for liquid chromatography mass spectrometry. TLC indicated that diacylglycerol (DAG) may be present in the tear film. PLC was in the tear film with an activity determined at approximately 15 mU/mL, equivalent to the removal of head groups from phosphatidylcholine at a rate of approximately 15 µM/min. Conclusions: This work shows that phospholipid was not detected in any of the tested human tear samples (above the lower limits of detection as described) and suggests the presence of DAG in the tear film. DAG is known to be at low concentrations in MGS. These observations indicate that PLC may play a role in modulating the tear film phospholipid concentration.

Ageing, adipose tissue, fatty acids and inflammation

A common feature of ageing is the alteration in tissue distribution and composition, with a shift in fat away from lower body and subcutaneous depots to visceral and ectopic sites. Redistribution of adipose tissue towards an ectopic site can have dramatic effects on metabolic function. In skeletal muscle, increased ectopic adiposity is linked to insulin resistance through lipid mediators such as ceramide or DAG, inhibiting the insulin receptor signalling pathway. Additionally, the risk of developing cardiovascular disease is increased with elevated visceral adipose distribution. In ageing, adipose tissue becomes dysfunctional, with the pathway of differentiation of preadipocytes to mature adipocytes becoming impaired; this results in dysfunctional adipocytes less able to store fat and subsequent fat redistribution to ectopic sites. Low grade systemic inflammation is commonly observed in ageing, and may drive the adipose tissue dysfunction, as proinflammatory cytokines are capable of inhibiting adipocyte differentiation. Beyond increased ectopic adiposity, the effect of impaired adipose tissue function is an elevation in systemic free fatty acids (FFA), a common feature of many metabolic disorders. Saturated fatty acids can be regarded as the most detrimental of FFA, being capable of inducing insulin resistance and inflammation through lipid mediators such as ceramide, which can increase risk of developing atherosclerosis. Elevated FFA, in particular saturated fatty acids, maybe a driving factor for both the increased insulin resistance, cardiovascular disease risk and inflammation in older adults.

(Table 2) Collection sites and number of specimens of Usnea subgenus Neuropogon species

Usnea species of the Neuropogon group are amongst the most widespread and abundant macrolichens in Antarctic regions. Four principal species, U. antarctica, U. aurantiaco-atra, U. sphacelata and U. subantarctica, have been described on morphological grounds. However, identification to species level is often difficult and atypical morphologies frequently arise. Over 400 specimens were collected on the Antarctic Peninsula and Falkland Islands. Both morphological and molecular characters (ITS and RPB1) were used to compare samples to clarify taxonomic relationships. Morphological characteristics used included presence of apothecia, apothecial rays, soredia, papillae, fibrils, pigmentation and the diameter of the central axis as a proportion of branch diameter. Results revealed a very close relationship between U. antarctica and U. aurantiaco-atra, suggesting that they might constitute a species pair or be conspecific. Usnea sphacelata was comprised of at least two genetically distinct groups with no clear differences in morphology. One group included the first reported fertile specimen of this species. Usnea subantarctica was phylogenetically distinct from the other main Antarctic Usnea species, but clustered with U. trachycarpa. Genetic variation was evident within all species although there was no clear correlation between geographic origin and genetic relatedness. Phylogenetic analyses indicated that species circumscription in the Neuropogon group needs revision, with the principal species being non-monophyletic. None of the morphological characters, or groups of characters, used in this study proved to be completely unambiguous markers for a single species. However, axis thickness was supported as being informative for the identification of monophyletic lineages within the group.

Primary data sets of the hydrographic atlas of the Southern Ocean

The general knowledge of the hydrographic structure of the Southern Ocean is still rather incomplete since observations particularly in the ice covered regions are cumbersome to be carried out. But we know from the available information that thermohaline processes have large amplitudes and cover a wide range of scales in this part of the world ocean. The modification of water masses around Antarctica have indeed a worldwide impact, these processes ultimately determine the cold state of the present climate in the world ocean. We have converted efforts of the German and Russian polar research institutions to collect and validate the presently available temperature, salinity and oxygen data of the ocean south of 30°S latitude. We have carried out this work in spite of the fact that the hydrographic programme of the World Ocean Circulation Experiment (WOCE) will provide more new information in due time, but its contribution to the high latitudes of the Southern Ocean is quite sparse. The modified picture of the hydrographic structure of the Southern Ocean presented in this atlas may serve the oceanographic community in many ways and help to unravel the role of this ocean in the global climate system. This atlas could only be prepared with the altruistic assistance of many colleagues from various institutions worldwide who have provided us with their data and their advice. Their generous help is gratefully acknowledged. During two years scientists from the Arctic and Antarctic Research Institute in St. Petersburg and the Alfred Wegener Institute for Polar and Marine Research in Bremerhaven have cooperated in a fruitful way to establish the atlas and the archive of about 38749 validated hydrographic stations. We hope that both sources of information will be widely applied for future ocean studies and will serve as a reference state for global change considerations.

Age, body mass, mass change and dispersal distance of suckling and weaned grey seal (Halichoerus grypus) pups

Grey seal, Halichoerus grypus, pups in the breeding colony at Froan, Norway, have a bimodal pattern of early aquatic behaviour. About 40% of the pups spend their time ashore to save energy, which can be allocated to growth or deposition of energy-rich adipose tissue. The other 60% of the pups enter the sea during suckling and the early postweaning period, and disperse to other locations within the breeding colony. Pups may swim distances up to 12 km. Neonatal aquatic dispersal behaviour may lead to increased energy expenditure for thermoregulation and swimming, and thus lead to a low rate of body mass gain during suckling and a high rate of body mass loss after weaning. Thus, we examined relationships between natal aquatic dispersal behaviour and change in body mass (DeltaBM) in suckling and weaned pups. Suckling pups that had dispersed >2000 m had a significantly lower DBM than suckling pups that dispersed <2000 m or that did not disperse. In weaned pups, there were no effects of aquatic dispersal behaviour on DBM. We suggest that the bimodal natal aquatic dispersal behaviour in grey seals at the study site reflects two different strategies for postweaning survival: to stay ashore and get fat, or to take a swim and acquire diving and feeding skills.

De Rol van Sekse in de Relatie tussen Sociale Stress, Fysieke Activiteit en Negatief Affect bij Adolescenten

Diverse onderzoeken bij adolescenten tonen, met name bij meisjes, een verband aan tussen sociale stress, fysieke activiteit en negatief affect en het ontwikkelen van depressie. Jongeren kunnen tijdens de adolescentiefase meer negatief affect ervaren als gevolg van te veel sociale stress en te weinig fysieke activiteit. In dit onderzoek is specifiek gekeken naar het verband tussen sociale stress en negatief affect en naar het verband tussen fysieke activiteit en negatief affect en naar de rol van sekse in deze verbanden. De data zijn verzameld middels de Experience Sampling Methode (ESM); via een applicatie op de smartphone vulden 33 adolescenten tussen de 12 en 18 jaar gedurende vijf dagen (drie weekdagen en twee weekenddagen) op 10 willekeurige tijdstippen per dag vragenlijsten in met betrekking tot sociale stress, fysieke activiteit en negatief affect. Multilevel regressie analyse toont aan dat er een significant positief verband is tussen sociale stress en negatief affect en dat sekse dit verband niet modereert. In dit onderzoek wordt geen relatie aangetoond tussen fysieke activiteit en negatief affect. Vervolgonderzoek naar determinanten van sociale stress, fysieke activiteit en de rol van sekse is nodig om meer inzicht te krijgen in risico- en protectieve factoren ten aanzien van het ontwikkelen van depressie.

Analysis of the signal transduction in the hawk moth Manduca sexta

Fliegende Insekten orientieren sich in ihrer Umwelt mit Hilfe ihres hoch entwickelten olfaktorischen Systems. Es ermöglicht ihnen das Auffinden geeigneter Futter- und Eiablageplätze und ist unverzichtbar bei der innerartlichen Kommunikation. Der Geruchssinn muss dabei gleichzeitig sehr schnell und sensitiv sein um selbst geringste Mengen, z.B. des arteigenen Sexualpheromons, wahrnehmen zu können. Spezifische olfaktorische Rezeptoren (ORs) zur Detektion dieser Duftstoffe werden zusammen mit einem hoch konservierten Co-Rezeptor (Orco) in olfaktorischen Rezeptorneuronen (ORNs) auf den Insektenantennen exprimiert. Sie gehören zu den 7 Transmembran Rezeptoren, zeigen jedoch eine invertierte Membrantopologie im Vergleich zu den ORs der Vertebraten. Darüber hinaus bildet der OR/Orco-Komplex einen spontanaktiven Kationenkanal, die Bindung an ein G Protein ist allerdings umstritten. Daher ist noch ungeklärt, ob die Duftstoffbindung zu einer ionotropen Aktivierung des OR/Orco Kanals führt oder ob metabotrope Mechanismen die Bildung von zyklischem Adenosinmonophosphat (cAMP) oder Inositol 1,4,5-trisphosphat (IP3) bewirken. Mit Hilfe von extrazellulären Ableitungen einzelner Trichoidsensillen (tip recordings) auf den Antennen männlicher Manduca sexta wurde die Rolle von Orco sowie die Beteiligung einer Phospholipase Cβ (PLCβ)-abhängigen Transduktionskaskade untersucht. Es konnte gezeigt werden, dass die durch VUAA1 induzierte Spontanaktivität der ORNs durch OLC15 inhibiert und Orco somit kompetitiv gehemmt wurde. Eine Inhibition von Orco sollte die Antwort auf kurze Pheromonpulse sofort reduzieren, sollte die Transduktion über die Aktivierung des OR/Orco Kanals erfolgen. Die Ergebnisse dieser Arbeit zeigten jedoch keine Beeinflussung der primären Pheromonantwort, vielmehr wurde die späte, langanhaltende Antwort reduziert. Die ebenfalls als Orco-Antagonisten charakterisierten Amiloride MIA und HMA beeinflussen offensichtlich weitere Ziele, da eine substanz- und zeitgeberzeitabhängige Reduzierung der primären Antwort auftrat. Zusätzlich wurde die primäre Pheromonantwort durch die Inhibierung der PLCβ und der Proteinkinase C (PKC), sowie durch die Verwendung zweier Diacylglycerol (DAG)- Derivate signifikant beeinflusst. Hierbei zeigte die Inhibierung von PLCβ und PKC zeitgeberzeitabhängige Unterschiede in der Stärke der Antwortreduktion. Auch die Applikation des DAG-Derivates DOG reduzierte die Pheromonantwort, während die Zugabe von OAG die ORN Aktivität steigern oder reduzieren konnte, abhängig von der verwendeten Derivatkonzentration und der Pheromonkonzentration. Die Ergebnisse dieser Arbeit deuten somit auf einen metabotropen, sehr wahrscheinlich PLCβ-abhängigen Mechanismus für die Pheromontransduktion bei Manduca sexta.

Störningsinformation i tågtrafiken : Attityder bland resenärer och tågbolag till störningsinformation och tjänster för sådan information

When booking a trip along the railway through several train operators it is not uncommon that information about possible disruptions along the railway (that can change or cancel the booked trip) are not relayed to the passengers. Today, research on rail traffic in Sweden is limited. It is unclear how satisfied customers are with the quality of the information they receive during their trip (if they get it at all), including with respect to disruptions. Our partners have identified what they believe is a need among train operators, which is a service for disruption information to travelers. In addition to confirming that there is a need for such a service, we have an interest to investigate how such a service might look like and what the users want. Our research has shown that passengers are not satisfied with either the amount of information about disturbances or how often they get it. Along with KnowitBorlänge, we have come up with a proposed solution that uses already existing technologies to create a portal for an efficient way to get the interference information to travelers.

Quantification of upper limb motor symptoms of Parkinson’s disease using a smartphone

Panda

Extramural engelska : En tillgång i engelskundervisningen?

Under de senaste tio åren har den digitala teknikens omfattning ökat och är idag en källa där barn och unga möter engelska i princip varje dag. Många elever använder dagligen engelska i tal och skrift för olika mottagare och syften genom bland annat datorspel och sociala nätverk. Syftet med denna litteraturstudie är således att granska extramural engelska och vad det kan tillföra engelskundervisningen i skolan. Med extramural engelska menas all den engelska en elev kommer i kontakt med utanför skolan. Studien är begränsad till att omfatta elever i årskurs 4-9, eftersom forskningsfältet är begränsat i de lägre årskurserna 4-6 behövdes även årskurs 7-9 inkluderas. Materialet består av vetenskapliga publikationer och samlades in genom fyra söktjänster som behandlar vetenskaplig litteratur. Resultatet visar att extramural engelska har en påverkan på elevernas olika språkliga förmågor. Det finns även vissa kopplingar mellan elevernas extramurala engelska och deras resultat på nationella prov och betyg i ämnet.  Kopplingen mellan elevernas extramurala engelska och deras olika språkliga förmågor är betydligt starkare än kopplingen mellan extramural engelska och elevernas betyg i ämnet engelska. Ett flertal av de studier vi analyserat är överens om att elever som ägnar mer tid åt extramural engelska generellt har ett bättre betyg, men att det inte går att dra någon generell slutsats att det endast är extramural engelska som påverkar betyget. Vad gäller elevernas olika språkliga förmågor påverkar extramural engelska främst elevernas vokabulär men även läs- och hörförståelse samt muntlig och skriftlig förmåga.

Débalancement du métabolisme des acides gras polyinsaturés à longues chaînes chez les porteurs de l'apolipoprotéine E ε4

Les porteurs de l’apolipoprotéine E ε4 (APOE4) sont à risque accru de développer un déclin cognitif et/ou des maladies cardiovasculaires comparativement aux non-porteurs. Ceci serait partiellement attribuable à un débalancement dans le métabolisme de l’acide docosahexaénoïque (DHA), un acide gras (AG) polyinsaturé oméga-3 qui joue un rôle crucial dans la santé du cerveau et du cœur. La consommation d’une diète riche en AG saturés et la présence de surpoids pourraient exacerber ce débalancement puisque ces facteurs modifient l’homéostasie du DHA. Des données préliminaires suggèrent que la consommation d’un supplément à haute dose de DHA, sur le long terme, permettrait de rétablir l’homéostasie de cet AG chez les porteurs de l’APOE4. L’objectif de la première étude était d’évaluer la réponse plasmatique à un supplément de DHA chez des participants consommant une diète riche en AG saturés, et ce, en fonction de l’indice de masse corporelle (IMC) et du statut de porteur de l’APOE4. Cette étude a démontré une interaction génotype x IMC sur la réponse plasmatique de l’acide arachidonique (AA) et du DHA au supplément. De plus, les porteurs de l'APOE4 étaient de plus faibles répondeurs au supplément de DHA comparativement aux non-porteurs, mais seulement chez les participants en surpoids. L’objectif de la seconde étude était d’évaluer si une diète riche en DHA pendant huit mois permet de rétablir les niveaux de DHA dans le foie de souris porteuses de l’APOE4 et d’évaluer si les transporteurs hépatiques d’AG sont impliqués dans ce rétablissement. Les résultats ont démontré que sous une diète contrôle, les concentrations hépatiques d’AA et de DHA étaient plus élevées chez les souris APOE4 comparativement aux souris APOE3 et que le transporteur d’AG hépatiques FABP1 est impliqué. Par contre, chez les souris ayant consommé la diète riche en DHA, les niveaux d’AA et de DHA étaient similaires entre les génotypes. Ceci suggère qu’une supplémentation à long terme en DHA pourrait rétablir l’homéostasie de l’AA et du DHA chez les porteurs de l’APOE4. Puisque le DHA est impliqué dans la santé du cerveau et du cœur, la consommation de hautes doses de DHA chez les porteurs de l’APOE4 pourrait contribuer à diminuer leur risque de développer un déclin cognitif et/ou des maladies cardiovasculaires mais cette association devra être évaluée dans des études ultérieures.